A Pol V–Mediated Silencing, Independent of RNA–Directed DNA Methylation, Applies to 5S rDNA

The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA–directed DNA methylation (RdDM), which also requires activities from RDR2 (RNA–Dependent RNA Polymerase 2), DCL3 (Dicer-Like 3), AGO4 (Argonaute), and DRM2 (Domains Rearranged Methyltransferase 2). RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA–encoding DNA (rDNA) arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V–loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM–independent and comes specifically at chromosome 4, in addition to the RdDM pathway

Oxidative stress, Nrf2 and keratin up‐regulation associate with Mallory‐Denk body formation in mouse erythropoietic protoporphyria

Mallory‐Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech m1Pas mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC‐induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug‐induced MDBs. In 13‐ and 20‐week‐old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13‐week‐old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase‐2 and keratin overexpression, with a greater keratin 8 (K8)‐to‐keratin 18 (K18) ratio, which are critical for drug‐induced MDB formation. In 20‐week‐old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short‐term (3‐week) DDC feeding markedly induced MDB formation in 20‐week‐old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative‐stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up‐regulation. Nrf2 knockdown in HepG2 cells down‐regulated K8, but not K18. Conclusion : Fch/fch mice develop age‐associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2‐mediated increase in K8, promotes MDB formation. (H epatology 2012;56:322–331)Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/92074/1/25664_ftp.pd

The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants

​The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex is an evolutionary well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data support its function in nuclear morphology and meiosis, its implication in chromatin organisation has not been studied in plants. Here 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the model plant Arabidopsis thaliana, in which heterochromatin cluster in conspicuous chromatin domains called chromocentres. Chromocentres form a repressive chromatin environment contributing to transcriptional silencing of repeated sequences, a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres indicate their close proximity to the nuclear periphery but that their position varies with nuclear volume and can be altered in specific mutants affecting the LINC complex. Finally we propose that the plant LINC complex contributes to proper heterochromatin organisation and positioning at the nuclear periphery, since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences

L'induction de mutations chez la souris. Production de modeles animaux. Interet pour l'etude du developpement

SIGLEINIST T 74995 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Analysis of 5S rDNA Loci among Arabidopsis Ecotypes and Subspecies

International audienc

An analysis of retroposition in plants based on a family of SINEs from Brassica napus

International audienceThe identification of a family of SINE retroposons dispersed in the genome of oilseed rape Brassica napus has provided the basis for an evolutionary analysis of retroposition in plants. The repetitive elements (called S1Bn) are 170 bp long and occupy roughly 500 loci by haploid genome. They present characteristic features of SINE retroposons such as a 3' terminal A-rich region, two conserved polymerase III motifs (box A and B), flanking direct repeats of variable sizes, and a primary and secondary sequence homology to several tRNA species. A consensus sequence was made from the alignment of 34 members of the family. The retroposon population was divided into five subfamilies based on several correlated sets of mutations from the consensus. These precise separations in subfamilies based on "diagnostic" mutations and the random distribution of mutations observed inside each subfamily are consistent with the master sequence model proposed for the dispersion of mammalian retroposons. An independent analysis of each subfamily provides strong evidence for the coexpression of at least three subfamily master sequences (SMS). In contrast to mammalian retroposition, diagnostic positions are not shared between SMS. We therefore propose that SMS were all derived from a general master sequence (GMS) and independently activated for retroposition after a variable period of random drift. Possible models for plant retroposition are discussed

Athila, a new retroelement from Arabidopsis thaliana.

International audienceAn analysis of Arabidopsis thaliana heterochromatic regions allowed the identification of a new family of retroelements called Athila. These 10.5 kb elements, representing ca. 0.3% of the genome, present several features of retrotransposons and retroviruses. Athila elements are flanked by 1.5 kb long terminal repeats (LTR) that are themselves bounded by 5 bp perfect inverted repeats. These LTRs start and end with the retroviral consensus 5'TG...CA3' nucleotides. A putative tRNA-binding site and a polypurine tract are found adjacent to the 5' and 3' LTR respectively. The central domain is composed of two long open reading frames (ORFs) of 935 and 694 amino acids. Despite several indications of recent transposition activity, the translation of these ORFs failed to reveal significant homology with proteins associated to retrotransposition. We suggest that the Athila family could result from the transduction and dispersion of a cellular gene by a retrotransposon

Characterization of two distinct subfamilies of SUN-domain proteins in Arabidopsis and their interactions with the novel KASH-domain protein AtTIK

International audienceSUN-domain proteins belong to a gene family including classical Cter-SUN and mid-SUN subfamilies differentiated by the position of the SUN domain within the protein. Although present in animal and plant species, mid-SUN proteins have so far remained poorly described. Here, we used a combination of genetics, yeast two-hybrid and in planta transient expression methods to better characterize the SUN family in Arabidopsis thaliana. First, we validated the mid-SUN protein subfamily as a monophyletic group conserved from yeast to plant. Arabidopsis Cter-SUN (AtSUN1 and AtSUN2) and mid-SUN (AtSUN3 and AtSUN4) proteins expressed as fluorescent protein fusions are membrane-associated and localize to the nuclear envelope (NE) and endoplasmic reticulum. However, only the Cter-SUN subfamily is enriched at the NE. We investigated interactions in and between members of the two subfamilies and identified the coiled-coil domain as necessary for mediating interactions. The functional significance of the mid-SUN subfamily was further confirmed in mutant plants as essential for early seed development and involved in nuclear morphology. Finally, we demonstrated that both subfamilies interact with the KASH domain of AtWIP1 and identified a new root-specific KASH-domain protein, AtTIK. AtTIK localizes to the NE and affects nuclear morphology. Our study indicates that Arabidopsis Cter-SUN and mid-SUN proteins are involved in a complex protein network at the nuclear membranes, reminiscent of the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex found in other kingdoms

DNA regions flanking the major Arabidopsis thaliana satellite are principally enriched in Athila retroelement sequences.

International audienceAn analysis of Arabidopsis thaliana heterochromatic regions revealed that genomic sequences immediately flanking the major 180 bp satellite are essentially made of middle repetitive sequences and that most of these sequences correspond to defective Athila retroelements. Using YAC and lambda clones, we evaluated the distribution of Athila elements in the Arabidopsis genome and showed that, despite the presence of numerous euchromatic copies, these elements are especially concentrated in or near heterochromatic regions. Sequencing of the various DNA transitions between satellite and Athila repeats provides strong evidence that most of the heterochromatic elements retrotransposed directly into 180 bp satellite clusters